Preparing Mtb Secretome to Study Host-Pathogen Interactions

<ol>
	<li>Campbell, I. A., Bah-Sow, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+tuberculosis:+diagnosis+and+treatment.">Pulmonary tuberculosis: diagnosis and treatment.</a> Br Med J. 332 (7551), 1194-1197 (2006).</li><li>Sonnenberg, M. G., Belisle, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition+of+Mycobacterium+tuberculosis+culture+filtrate+proteins+by+two-dimensional+polyacrylamide+gel+electrophoresis,+N-terminal+amino+acid+sequencing,+and+electrospray+mass+spectrometry.">Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry.</a> Infect Immun. 65 (11), 4515-4524 (1997).</li><li>Burts, M. L., Williams, W. A., DeBord, K., Missiakas, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EsxA+and+EsxB+are+secreted+by+an+ESAT-6-like+system+that+is+required+for+the+pathogenesis+of+Staphylococcus+aureus+infections.">EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections.</a> Proc Natl Acad Sci U S A. 102 (4), 1169-1174 (2005).</li><li>Braunstein, M., Brown, A. M., Kurtz, S., Jacobs, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+nonredundant+SecA+homologues+function+in+Mycobacteria.">Two nonredundant SecA homologues function in Mycobacteria.</a> J Bacteriol. 183 (24), 6979-6990 (2001).</li><li>Feltcher, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+quantitative+proteomics+reveals+a+role+for+the+Mycobacterium+tuberculosis+SecA2+pathway+in+exporting+solute+binding+proteins+and+Mce+transporters+to+the+cell+wall.">Label-free quantitative proteomics reveals a role for the Mycobacterium tuberculosis SecA2 pathway in exporting solute binding proteins and Mce transporters to the cell wall.</a> Mol Cell Proteomics. 14 (6), 1501-1516 (2015).</li><li>McDonough, J. A., Hacker, K. E., Flores, A. R., Pavelka, M. S., Braunstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+twin-arginine+translocation+pathway+of+Mycobacterium+smegmatis+is+functional+and+required+for+the+export+of+mycobacterial+beta-lactamases.">The twin-arginine translocation pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases.</a> J Bacteriol. 187 (22), 7667-7679 (2005).</li><li>Saint-Joanis, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+Rv2525c,+a+substrate+of+the+twin+arginine+translocation+(Tat)+system+of+Mycobacterium+tuberculosis,+increases+beta-lactam+susceptibility+and+virulence.">Inactivation of Rv2525c, a substrate of the twin arginine translocation (Tat) system of Mycobacterium tuberculosis, increases beta-lactam susceptibility and virulence.</a> J Bacteriol. 188 (18), 6669-6679 (2006).</li><li>Braunstein, M., Espinosa, B. J., Chan, J., Belisle, J. T., Jacobs, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SecA2+functions+in+the+secretion+of+superoxide+dismutase+A+and+in+the+virulence+of+Mycobacterium+tuberculosis.">SecA2 functions in the secretion of superoxide dismutase A and in the virulence of Mycobacterium tuberculosis.</a> Mol Microbiol. 48 (2), 453-464 (2003).</li><li>Zulauf, K. E., Sullivan, J. T., Braunstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SecA2+pathway+of+Mycobacterium+tuberculosis+exports+effectors+that+work+in+concert+to+arrest+phagosome+and+autophagosome+maturation.">The SecA2 pathway of Mycobacterium tuberculosis exports effectors that work in concert to arrest phagosome and autophagosome maturation.</a> PLoS Pathog. 14 (4), e1007011 (2018).</li><li>Guinn, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+RD1-region+genes+are+required+for+export+of+ESAT-6/CFP-10+and+for+virulence+of+Mycobacterium+tuberculosis.">Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis.</a> Mol Microbiol. 51 (2), 359-370 (2004).</li><li>Lewis, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+RD1+from+Mycobacterium+tuberculosis+mimics+bacille+Calmette-Guerin+attenuation.">Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guerin attenuation.</a> J Infect Dis. 187 (1), 117-123 (2003).</li><li>Kana, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+resuscitation-promoting+factors+of+Mycobacterium+tuberculosis+are+required+for+virulence+and+resuscitation+from+dormancy+but+are+collectively+dispensable+for+growth+in+vitro.">The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro.</a> Mol Microbiol. 67 (3), 672-684 (2008).</li><li>Russell-Goldman, E., Xu, J., Wang, X., Chan, J., Tufariello, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mycobacterium+tuberculosis+Rpf+double-knockout+strain+exhibits+profound+defects+in+reactivation+from+chronic+tuberculosis+and+innate+immunity+phenotypes.">A Mycobacterium tuberculosis Rpf double-knockout strain exhibits profound defects in reactivation from chronic tuberculosis and innate immunity phenotypes.</a> Infect Immun. 76 (9), 4269-4281 (2008).</li><li>Biketov, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+resuscitation+promoting+factors+in+pathogenesis+and+reactivation+of+Mycobacterium+tuberculosis+during+intra-peritoneal+infection+in+mice.">The role of resuscitation promoting factors in pathogenesis and reactivation of Mycobacterium tuberculosis during intra-peritoneal infection in mice.</a> BMC Infect Dis. 7, 146 (2007).</li><li>Verma, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomic+and+phosphoproteomic+analysis+of+H37Ra+and+H37Rv+strains+of+Mycobacterium+tuberculosis.">Quantitative proteomic and phosphoproteomic analysis of H37Ra and H37Rv strains of Mycobacterium tuberculosis.</a> J Proteome Res. 16 (4), 1632-1645 (2017).</li><li>Malakar, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+CFP10+modulates+Mycobacterium+tuberculosis.">Phosphorylation of CFP10 modulates Mycobacterium tuberculosis.</a> mBio. 14 (5), e0123223 (2023).</li><li>Lochab, S., Singh, Y., Sengupta, S., Nandicoori, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+exploits+host+ATM+kinase+for+survival+advantage+through+SecA2+secretome.">Mycobacterium tuberculosis exploits host ATM kinase for survival advantage through SecA2 secretome.</a> Elife. 9, e51466 (2020).</li><li>Kurtz, S., McKinnon, K. P., Runge, M. S., Ting, J. P., Braunstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SecA2+secretion+factor+of+Mycobacterium+tuberculosis+promotes+growth+in+macrophages+and+inhibits+the+host+immune+response.">The SecA2 secretion factor of Mycobacterium tuberculosis promotes growth in macrophages and inhibits the host immune response.</a> Infect Immun. 74 (12), 6855-6864 (2006).</li><li>Sullivan, J. T., Young, E. F., McCann, J. R., Braunstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mycobacterium+tuberculosis+SecA2+system+subverts+phagosome+maturation+to+promote+growth+in+macrophages.">The Mycobacterium tuberculosis SecA2 system subverts phagosome maturation to promote growth in macrophages.</a> Infect Immun. 80 (3), 996-1006 (2012).</li><li>Putim, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretome+profile+analysis+of+multidrug-resistant,+monodrug-resistant+and+drug-susceptible+Mycobacterium+tuberculosis.">Secretome profile analysis of multidrug-resistant, monodrug-resistant and drug-susceptible Mycobacterium tuberculosis.</a> Arch Microbiol. 200 (2), 299-309 (2018).</li><li>Naz, S., Dabral, S., Kumar, D., Nandicoori, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+ex+vivo+competition+and+sequencing+of+Mycobacterium+isolated+from+infected+guinea+pigs.">Protocol for ex vivo competition and sequencing of Mycobacterium isolated from infected guinea pigs.</a> STAR Protoc. 3 (4), 101804 (2022).</li><li>Xu, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+identification+of+serum+potential+biomarkers+for+pulmonary+tuberculosis+using+iTRAQ-coupled+two-dimensional+LC-MS/MS.">Discovery and identification of serum potential biomarkers for pulmonary tuberculosis using iTRAQ-coupled two-dimensional LC-MS/MS.</a> Proteomics. 14 (2-3), 322-331 (2014).</li><li>Prasad, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+purified+protein+derivative+of+Mycobacterium+tuberculosis.">Proteomic analysis of purified protein derivative of Mycobacterium tuberculosis.</a> Clin Proteomics. 10 (1), 8 (2013).</li><li>Miller, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+SatS+is+a+chaperone+for+the+SecA2+protein+export+pathway.">Mycobacterium tuberculosis SatS is a chaperone for the SecA2 protein export pathway.</a> Elife. 8, e40063 (2019).</li></ol>

The authors have nothing to disclose.

One of the critical research areas pertains to understanding Mtb pathogenesis and developing effective diagnostics and vaccines22,23. Isolation of culture filtrate proteins from Mtb culture media is a process that enables the identification of the virulence factors secreted by the pathogen. Investigating the role of secretory proteins in modulating Mtb pathogenesis yields mechanistic insights and may also provide candidates for therapeu...

Mycobacterium tuberculosis (Mtb), one of the deadliest pathogens worldwide, infects healthy individuals through aerosols transmitted from an infected person while sneezing or coughing1. Once it enters the lungs, it is engulfed by the alveolar macrophages, hijacking the host immune system to survive. Among various host modulatory mechanisms that Mtb has for survival, secretory virulence factors are one of them. The proteins secreted into the culture media by the bacilli during its growth are termed Culture Filtrate (CF) proteins2. These culture filtrate proteins help bacteria to survive in harsh conditions inside the host cells. Mtb is equipped with three different kinds of secretion systems: a specialized Type VII secretion system (T7SS), a general Sec secretion system (Sec system), and a twin-arginine secretion system (Tat system). T7SS is the most exclusive secretion system in actinobacteria, unlike type I-VI in Gram-negative bacteria3. The secretion of unfolded protein across the membrane is majorly involved in the Sec system, and the proteins are recognized by an N-terminal signal sequence4,5. Folded or partially folded proteins are transported through the Tat system, identified by an N-terminal signal sequence with a twin-arginine motif6,7. How the Sec and Tat system regulates protein secretion has yet to be determined.
Some known culture filtrate proteins in Mtb that help withstand and survive the host oxidative onslaught upon infection are enzymes such as KatG and SodA8,9, necessary for degrading reactive oxygen species. In addition, CFP10-ESAT6, a heterodimeric protein complex, plays a vital role in regulating cytokine responses, bacterial escape, ROS production, etc.10. RD1 locus is involved in modulating the Type VII secretion system, which includes secretion of CFP10-ESAT6, and the genetic deletion of cpf10-esat6 compromises the survival of bacteria in ex vivo and in vivo infection models. Mycobacterium bovis BCG vaccine strain lacks RD1 locus responsible for the secretion of virulence factors and hence can be used as the live attenuated vaccine11. Different unexplored cellular processes tightly regulate the secretion process. The culture filtrate proteins contain resuscitation-promoting factors (RpFs) that help the bacteria regrow from the latent state. Resuscitation-promoting factors (Rpfs) are peptidoglycan hydrolyzing enzymes that are known to play a role in dormancy. Mtb encodes for five rpf-like genes (rpf A-E) that help the bacteria regrow from dormancy either solo or by interacting with other proteins12. Rpfs are found in bacterial culture filtrate and are known to be involved in Mtb virulence and host immunity13,14. The widely studied rpf genes rpfE and rpfB show protective immunity in vitro13 and enhance immune responses in vivo. Hence, rpfE and rpfB can be used as Mtb vaccine candidates.
Reversible phosphorylation15 is a post-translational modification that alters various cellular processes, cell homeostasis, cell division, etc. For example, phosphorylation of CFP10 plays an important regulatory role, where both phosphoablative and mimetic mutants of CFP10 fail to restore the observed phenotype in the CFP10-ESAT6 deletion strain16. SecA2, on the other hand, is a secretory protein involved in DNA damage of the host cell17. The secretome of the SecA2 mutant is sufficient to change the host immune response and signaling cascade18,19. Thus, it is essential and exciting to investigate the secretome of H37Rv and the Mtb mutants, where the genes of the secretory proteins are deleted (unpublished data), to gain insights into how they impact host immunomodulation and survival.
This report focuses on three aspects: the steps involved in preparing Mtb culture filtrate, processing the samples for mass spectrometry, and performing ex vivo experiments with culture filtrates to examine immunomodulatory functions. Earlier studies showed the identification of culture filtrate proteins by 2D gel electrophoresis followed by Mass spectrometric analysis8. Culture filtrate proteins from drug-sensitive and resistant strains were identified using mass spectrometry to gain insights into the secretome changes and identify virulence factors that may play a role in drug resistance20. We have previously investigated Mtb&#39;s secretome and phosphosecretome with the help of mass spectrometry16. Techniques such as 2D gel electrophoresis are low throughput and are not used to investigate global changes in the proteome or secretome. The culture filtrate proteins would vary from bacterial strains, stages of the growing cells, and even in various stress conditions. Depending on different situations, the change in the secretory protein pools inside the cells would offer to explore the whole secretome holistically and provide more information about the bacterial secretion system for therapeutic interventions. It would be even more attractive to look at the secretome in the knockouts or depletion strains of secretory systems to gain further insights into the pathways and targets of each secretory pathway. Treating cells with culture filtrate proteins instead of bacteria changes cytokine levels and other immunomodulatory factors17. With the help of the secretome study, one can identify new virulence factors and their role in virulence.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#181;m Syringe filters</td>
			<td>mdi</td>
			<td>SYPL0601MNXX204</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2&#181;m Nitrocellulose membrane</td>
			<td>Amersham</td>
			<td>10600001</td>
			<td></td>
		</tr>
		<tr>
			<td>37% HCl</td>
			<td>Merck</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>EMPLURA MERCK</td>
			<td>SL9F690866</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonytrile</td>
			<td>Sigma-Aldrich</td>
			<td>34851-2L</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>BIORAD</td>
			<td>1610107</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Bio Basic Canada</td>
			<td>Cat No: AD0023</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15</td>
			<td>Merck, Millipore</td>
			<td>UFC900396</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium biocarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>09830-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulpahte</td>
			<td>Sigma-Aldrich</td>
			<td>A3678-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Ag85B</td>
			<td>Abcam</td>
			<td>AB43019</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse HRP conjugated secondary antibody</td>
			<td>Thermo Fischer</td>
			<td>62-6520</td>
			<td></td>
		</tr>
		<tr>
			<td>Asparagine</td>
			<td>Sigma-Aldrich</td>
			<td>A0884-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>bis-acrylamide</td>
			<td>SIGMA</td>
			<td>146072-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Merck</td>
			<td>MB083-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>C-18 column</td>
			<td>Thermo Fischer</td>
			<td>SEM SS18V</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C1345</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>C0759-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Middle brook 7H9 broth&#160;&#160;</td>
			<td>Fisher Scientific</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferric ammonium(III) citrate</td>
			<td>Sigma-Aldrich</td>
			<td>F5879-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid 98-100%</td>
			<td>Merck</td>
			<td>Z0823704246</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid 100%</td>
			<td>Merck</td>
			<td>CD0C700160</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>SIGMA</td>
			<td>G8898-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilion Western Chemiluminescent HRP substrate</td>
			<td>Merck</td>
			<td>WBKLS0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Amersham</td>
			<td>RPN6302V</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulpahte</td>
			<td>Sigma-Aldrich</td>
			<td>M7506-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobroswer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Sigma</td>
			<td>Cat No: O1008</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA protein assay Kit</td>
			<td>Thermo Fischer</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Trypsin Protase</td>
			<td>Thermo Fischer</td>
			<td>90058</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteome Discoverer 2.0</td>
			<td colspan="2">Thermo Fischer Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Q-Exactive HF (Thermo Scientific) (proteomics experiments - peptide sequencing, protein identification and quantification)</td>
			<td colspan="2">Thermo Fischer Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>Cat No: S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7773-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulphate</td>
			<td>Sigma-Aldrich</td>
			<td>L3771-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Sigma-Aldrich</td>
			<td>C4706-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Temed</td>
			<td>Sigma-Aldrich</td>
			<td>T9281-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>BioRad</td>
			<td>161-0719</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>AMRESCO</td>
			<td>0234-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma-Aldrich</td>
			<td>P1754</td>
			<td></td>
		</tr>
		<tr>
			<td>UniProt protein data base</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>MP Bio</td>
			<td>194857</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of mycobacterium tuberculosis culture filtrate to understand tb pathogenesis

Mycobacterium tuberculosis (Mtb) secretes many proteins into the host to modulate various cellular processes, such as host signaling, immune response, apoptosis, etc., to enhance survival. While the function of a few secretory factors has been elucidated, many are yet to be characterized. Hence, investigating the Mtb secretory protein is an essential area of research. Mtb secretes proteins into the extracytoplasmic space with the help of three different secretion systems: general, twin-arginine, and specialized Type VII secretion system. One has to prepare the secretome to identify novel secretory molecules and functionally characterize the known factors. Here, we outline the method for preparing the secretome and sample preparation for mass spectrometry. Secretome preparation requires the utmost care to avoid contamination of cytosolic proteins, which can be monitored by probing with antibodies against well-characterized membrane, secretory, and cytosolic proteins. The culture filtrate thus prepared can be used for different purposes, which include investigating host immunomodulation properties.

In this protocol, we demonstrated the isolation of culture filtrate proteins from Mycobacteria (Figure 1A). The culture filtrate proteins were then checked for the different markers to confirm the culture filtrate isolation. As mentioned in the protocol, a Coomassie staining was performed to check the protein loading. One can observe a streaked protein band in the WCL fraction (at the last lane Figure 1B), whereas in CF (culture filtrate) lanes, all the...

Preparation of Mycobacterium Tuberculosis Culture Filtrate to Understand TB Pathogenesis

Here, we demonstrate the preparation of Mtb culture filtrate (CF), which is the bacterial growth media that consists of proteins secreted by the bacteria. Mtb secretes proteins to modulate host signaling and immune response, which are crucial for pathogenesis. The CFs prepared are analyzed by mass spectrometry to identify all the secretory factors.

Preparation of Mycobacterium tuberculosis Culture Filtrate to Understand TB Pathogenesis

Mycobacterium tuberculosis (Mtb) secretes many proteins into the host to modulate various cellular processes, such as host signaling, immune response, ...

preparation-mycobacterium-tuberculosis-culture-filtrate-to-understand

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

208 Views.  CSIR-Centre for Cellular and Molecular Biology (CSIR-CCMB). Here, we demonstrate the preparation of Mtb culture filtrate (CF), which is the bacterial growth media that consists of proteins secreted by the bacteria. Mtb secretes proteins to modulate host signaling and immune response, which are crucial for pathogenesis. The CFs prepared are analyzed by mass spectrometry to identify all the secretory factors.

Video: Preparation of Mycobacterium Tuberculosis Culture Filtrate to Understand TB Pathogenesis

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...